A method for improving the efficiency of CRISPR/Cas9-mediated biallelic mutation and its application

A gene-mediated technology, applied in the field of genetic engineering and molecular genetic breeding, can solve the problems affecting the efficiency of gene editing in animals such as the acquisition of target traits in animals, and achieve the effects of improving gene editing efficiency, increasing the ratio, and reducing the ratio of chimeric individuals

Active Publication Date: 2022-05-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The so-called "mosaic phenomenon" refers to the fact that when the CRISPR / Cas9 system is applied to the gene editing of fertilized eggs of multicellular organisms, since the fertilized eggs will split into different blastomeres, the editing ability and repair method of Cas9 protein on different blastomeres may be different, leading to the emergence of chimeric individuals with both edited cells and unedited cells, which in turn affects the efficiency of gene editing in animals and the acquisition of target traits in animals

Method used

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  • A method for improving the efficiency of CRISPR/Cas9-mediated biallelic mutation and its application
  • A method for improving the efficiency of CRISPR/Cas9-mediated biallelic mutation and its application
  • A method for improving the efficiency of CRISPR/Cas9-mediated biallelic mutation and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 sgRNA design for MSTN and FGF5 gene editing

[0039] In this example, the MSTN and FGF5 genes of sheep are used as examples to design sgRNA for CRISPR / Cas9 gene editing.

[0040] In order to achieve high gene editing efficiency, the design principles of sgRNA are as follows: the length of the sequence that is complementary to the genome sequence is 20-30nt, and there must be a PAM (NGG) sequence at the 3' end of the sequence, avoiding SNP and polyT sequences, It is best to start with the base "G".

[0041] Two targeting sites were designed for the MSTN gene of sheep, both located on the third exon of the MSTN gene. The arrangement and structure of the two targets in the MSTN genome are as follows figure 1 shown, named Cr1 and Cr2, figure 1The corresponding target sequence in the genome and the PAM segment of this site are shown. The gray sequence is the crRNA sequence corresponding to the sgRNA and the target site, followed by tracrRNA, which together const...

Embodiment 2

[0043] Example 2 In vitro transcription of Cas9 mRNA and sgRNA

[0044] Q5 high-fidelity DNA polymerase was used to amplify the Cas9 containing the T7 promoter (the Cas9 gene sequence is shown in SEQ ID NO. 4) and the sgRNA sequence, and after purification, the template transcribed in vitro was obtained. T7-Cas9 was transcribed with mMESSAGE mMachineT7Ultra Kit, and T7-sgRNA was transcribed with MEGAshortscript Kit. The transcribed Cas9 RNA becomes a single-stranded straight line after being treated with RNA-specific loading. After 2% agarose-TBE gel electrophoresis, it shows that the Cas9 cap is about 4200 nt after transcription, and the transcribed Cas9 mRNA band is clear and the size is correct (such as image 3 shown in A). The post-transcription sgRNA (about 100nt) was purified and recovered by agarose gel electrophoresis detection. The sgRNA band was clear and the size was correct (such as image 3 shown in B).

Embodiment 3

[0045] Example 3 Editing sheep MSTN and FGF5 genes using CRISPR / Cas9 system

[0046] In this example, the prokaryotic microinjection method was used to introduce the Cas9 mRNA and the sgRNAs of MSTN and FGF5 genes synthesized in vitro in Example 2 into sheep prokaryotic embryos to achieve gene knockout of MSTN and FGF5.

[0047] The Cas9 mRNA and sgRNA synthesized by in vitro transcription in Example 2 were mixed to prepare a gene editing nucleic acid solution for microinjection. According to the different injection ratios of Cas9 mRNA and sgRNA, sheep prokaryotic embryos were divided into three groups: (1) Control group: the molar concentration ratio of Cas9 mRNA and sgRNA was 1:2, a total of two microinjections were performed, and the first microinjection was performed. The concentrations of Cas9 mRNA and sgRNA were 5.23*10 -9 mol / l and 10.46*10 -9 mol / l, the concentrations of Cas9 mRNA and sgRNA in the second microinjection were 7.93*10 -9 mol / l and 15.86*10 -9 mol / l, t...

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Abstract

The invention relates to the technical fields of genetic engineering and molecular genetic breeding, in particular to a method for improving the efficiency of biallelic mutation mediated by CRISPR / Cas9 and its application. The CRISPR / Cas9 gene editing method provided by the present invention includes the step of introducing gene editing nucleic acid into the prokaryotic stage embryo, the gene editing nucleic acid including Cas9 mRNA and sgRNA; in the gene editing nucleic acid, the molar concentration of the Cas9 mRNA and sgRNA The ratio is 1:10~1:20. The gene editing method provided by the present invention significantly improves the efficiency of biallelic mutation, thereby effectively reducing the proportion of gene-edited chimeric individuals and increasing the proportion of biallelic mutant individuals. Breeding has important application value.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and molecular genetic breeding, in particular to a method for improving the efficiency of biallelic mutation mediated by CRISPR / Cas9 and its application. Background technique [0002] CRISPR / Cas9 technology is an RNA-mediated genome editing technology, which can precisely knock out, knock in and replace genes, so as to achieve the purpose of studying gene function, removing or repairing targeted genes. [0003] The CRISPR / Cas9 system mainly consists of two parts: sgRNA and Cas9 protein. sgRNA is composed of CRISPR RNA (crRNA) and trans-activating crRNA (trans-activating crRNA, tracrRNA). The crRNA includes sequences that can pair with tracrRNA to form a double-stranded RNA structure, and also include sequences that can be complementary to the target region of the target gene. This identifies the target sequence. In practical applications, crRNA and tracr RNA are often chimerized into...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/10A01K67/027
CPCC12N15/907C12N15/102A01K67/0275A01K2217/07A01K2227/103A01K2267/02
Inventor 连正兴李岩邓守龙刘国世连玲
Owner CHINA AGRI UNIV
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